Correlation between Apical Localization of Abcc2/Mrp2 and Phosphorylation Status of Ezrin in Rat Intestine
Correlation between Apical Localization of Abcc2/Mrp2 and Phosphorylation Status of Ezrin in Rat Intestine
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DOI：
10.1124/dmd.108.024836
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发表时间：
2009-07-01
影响因子：
3.9
通讯作者：
Horie, Toshiharu
中科院分区：
文献类型：
--
作者：
Nakano, Takafumi;Sekine, Shuichi;Horie, Toshiharu
多药耐药相关蛋白 2/ATP 结合盒转运蛋白家族 C2 (Mrp2/Abcc2) 是一种 ATP 依赖性输出泵，介导多种有机阴离子的转运。 Abcc2主要表达于肝细胞的小管膜和肠上皮细胞的刷状缘膜上。我们之前曾报道过，在急性氧化应激过程中，Abcc2 会迅速从肾小管膜内化，从而诱导大鼠肝脏中蛋白激酶 C (PKC) 激活。然而，尚未阐明PKC是否参与Abcc2在其他组织中定位的调节。在这项研究中，我们在大鼠肠上皮细胞中研究了这个问题。暴露于百里香毒素（一种传统的 PKC (cPKC) 激活剂）20 分钟，40 分钟内通过 Abcc2 的累积谷胱甘肽 S-bimane 流出量从 30.3 +/- 2.1 nmol/cm 降低至 18.1 +/- 1.6 nmol/cm。同样，小肠刷状缘膜中的 Abcc2 表达减少至对照的一半，而匀浆中存在的 Abcc2 总量没有改变。免疫沉淀分析表明 Abcc2 和 ezrin 之间存在相互作用，ezrin 是一种主要在肠道中表达的支架蛋白。胸腺毒素处理降低了埃兹蛋白活性形式（C 末端磷酸化形式）的量以及与埃兹蛋白共免疫沉淀的 Abcc2 的量。这些结果表明 cPKC 激活减弱了 ezrin 和 Abcc2 之间的蛋白质-蛋白质相互作用。总之，ezrin的磷酸化状态与大鼠小肠细胞表面Abcc2的表达相关，其可能受到cPKC的调节。
The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 +/- 2.1 nmol/cm to 18.1 +/- 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.